This invention relates to a process for separating and measuring two or more forms of glycoproteins which are different in sugar chain structure.
In not only human beings but also most animals, many of proteins contained in body fluids are glycoproteins having one or more sugar chains. Recently, because of the diversity of its structure and the like, the sugar chain moiety is particularly noted as a substance carrying some information concerning the interior of a living body and is studied in many universities and research institutes both inside and outside Japan.
In the course of the study, a change in the sugar chain structure of a specific glycoprotein caused by some disease was often observed. For example, in human .alpha.-fetoprotein (AFP), it was confirmed that the addition of an .alpha.-L-fucose residue or an N-acetylglucosamine residue (bisecting N-acetylglucosamine) to AFP sugar chain is frequently observed with the progress of hepatic cellular carcinoma. Furthermore, the degree of such a change of the sugar chain structure is independent of the AFP concentration in serum and the change is confirmed even in early hepatic cellular carcinoma. Therefore, it is considered that the investigation of the degree of the change permits diagnosis of cancer. Thus, the change of the sugar chain structure is noted.
The degree of change of the sugar chain structure of a glycoprotein is measured mainly by an analytical method using a lectin, such as lectin column method or lectin electrophoresis method. The reason why the degree of change of the sugar chain structure is measured mainly by use of a lectin is, for example, as follows. Lectins are not expensive. Moreover, since the immunogenicity of the sugar chain moiety of a glycoprotein is lower than that of the protein portion of the glycoprotein, no effective anti-sugar chain antibody can be obtained except that for a part of the non-reducing terminal structure, namely, the preparation of an antibody to the sugar chain of the glycoprotein is very difficult.
However, although lectins have a high recognizing specificity for the sugar chain structure, their bonding strength (coupling constant) to the sugar chain is one over thousands to one over tens of thousands as high as that of the antibody. Therefore, it is difficult to form a complex of a lectin and the sugar chain which is stable like a complex formed by antigen-antibody reaction. Accordingly, when a lectin is used in place of an antibody in an analytical method requiring a washing procedure after the formation of a complex on a solid phase, such as an enzyme immunoassay (ELISA) using a solidified antibody, a complex of the lectin and a glycoprotein to be measured dissociates during the washing, resulting in causing problems such as a marked lowering of the measuring sensitivity. Thus, such a method cannot be said to be practical as a clinical diagnosis. In addition, because of the above-mentioned properties of lectins, there has been the following restriction: for measuring a glycoprotein by utilizing the reaction of a lectin with the glycoprotein, the measurement should be carried out in the presence of a large excess of the lectin over the glycoprotein to be measured, by use of a column coupled with the lectin, an agarose gel containing the lectin, or the like.
Therefore, there is desired the development of a process for rapid and easy separation and measurement of glycoprotein sugar chains, which has both the sugar-recognizing specificity of lectins and the high sensitivity of enzyme immunoassay.